Real-Time PCR is a process of monitoring a PCR reaction by recording the fluorescence generated either by an intercalating dye such as SYBR Green or a target-specific reporter probe at each cycle. This is generally performed on a Real-Time PCR instrument that executes thermal cycling of the sample to complete the PCR cycles and at a specified point in each cycle measures the fluorescence of the sample in each channel through a series of excitation/emission filter sets.
In the age of whole genome sequencing and high-throughput analysis, there is a need for methods of detecting multiple qPCR targets at once. A limitation of current qPCR machines is the number of channels available for target detection. Modern instruments have between two and six channels, meaning that only two to six reporters can be detected in each experiment. Contemporary multiplexing technology provides for two to four different fluorescent reporters, and therefore two to four targets, to be detected in a single reaction. Unfortunately, accurately distinguishing each of the two to four targets from one another is thwarted by overlapping fluorescence signals of the different reporters. Therefore, a need still remains for a reliable and precise method for distinguishing multiple reporter signals during multiplex qPCR reactions.